Various techniques exist for the separation and extraction of target constituents from a specimen of a mixture of various constituent. The techniques are such that utilizes the physical properties derived from the molecular sizes, molecular weights, molecular shapes of the desired constituents, or biochemical properties such as solubility or affinity. For example, separation technology such as chromatography has been widely developed for the extraction of trace constituents contained in blood or other biological specimens. Development of separation techniques and optimization of methods using appropriate separation columns, solid-phase extraction packing sorbents, magnetic beads, etc., according to the target constituents, has been achieved. In addition, the volumes of specimens useable for preprocessing tend to become increasingly smaller, and in such tendency, a higher yield and more accurate separation/extraction are demanded. In accordance with these progress and advancement of separation/extraction techniques, a more efficient preprocessing system, that can meet the increase in the number of kinds of reaction solutions and steps used for separation/extraction, and is capable of separating/extracting target constituents from smaller quantities of specimens, is demanded.
The immunosuppressant drugs administered to organ transplant recipients, for example, tend to have high lipid-solubility and high partition ratio into blood cells. Therefore, upon measuring the concentration of an administered immunosuppressant drug in blood, it is necessary to preprocess by hemolyzing whole blood and removing the internal contents of the blood cells, and then extracting the drug adsorbed onto proteins or the like.
In general, hemolyzing can be executed by applying chemical, physical, or biological mechanisms. For example, hemolysis can be chemically caused by using various solvents or surface-active agents and thereby dissolving or damaging the lipids that constitute cell membranes. Applicable physical methods include pressurizing, centrifuging, mixing, freezing and thawing, hypotonizing (hypotensioning), and so on. Useable biological methods include forming transmembrane protein complexes by means of antibody binding or complement binding to the blood cells, forming holes/pores in the blood cell membranes using the hemolysins produced by pathogenic bacteria, and more.
Particularly, blood cell bursting by hypotonization is simple in principle. It is a method that ruptures cell membranes by reducing the salt concentration of blood with H2O (or the like) to reduce osmotic pressure around the blood cells, and thus causing excessive uptake of water into the blood cells. Normal saline solutions generally have a concentration equivalent to 0.90% NaCl, and it is known to cause hemolysis if diluted to a concentration equivalent to 0.50%-0.35% NaCl.
In addition, γ (gamma)-globulin and other key proteins present in large quantities in blood can be condensed and precipitated by applying the chelating effect of zinc. For this purpose, a method of conducting protein removal simultaneously with hemolyzing by adding a zinc sulfate solution, instead of H2O, to the blood is commonly used as an alternative method, which is also used for ZTT (Zinc sulfate Turbidity Testing) as well.
Another alternative method for collecting the desired constituents adsorbed onto the proteins in blood is deproteinization. Deproteinization is a process in which an organic solvent is added to a hemolyzed specimen to denature proteins and to extract desired constituents into the organic solvent. Centrifuging is generally provided to separate condensed proteins and a supernatant, and then to collect the supernatant. The deproteinization process is performed to condense and remove the proteins included in diverse forms and large volumes in blood. Conducting such process allows specimens derived from whole blood to be processed in the same manner as serum or plasma specimens.
As discussed above, organic solvents also yield a hemolytic effect. Therefore, hemolyzation and deproteinization may be conducted by adding an organic solvent to blood directly. Further, in order to complement the deproteinization effect in such cases, the foregoing method adding zinc sulfate may also be applied.
Performing the above-described process to a whole-blood specimen allows drugs with high blood cell partition ratio to be collected in a solution state. The specimen can then be subjected to purifying operations such as solid-phase extraction or liquid chromatographic separation. Typically, a supernatant collected after deproteinization is then dried up, and is redissolved in a solution of an appropriate volume so as to reduce the amount of liquid and to concentrate target constituents. After this, the redissolved solution is subjected to, for example, liquid chromatography-mass spectrometry (LCMS) or the like, whereby the concentrated target constituents are separated/purified and detected for subsequent identification and quantitative analysis.
The specimens from which the target constituents are separated and extracted using any one of those techniques are usually composed of various substances and constituents exhibiting different properties. To extract the desired trace constituents efficiently, the specimens need to undergo preprocessing such as specimen preparation and pre-purification to achieve an extractable state. Two basic technical elements used in common for such preprocessing are, a technique of dispensing solution and mixing the specimen with a reaction solution or the like by mixing, and a filtering technique. One technical problem associated with dispensing and mixing is carry-over problem during the preprocessing, where during continuous preprocessing of a plurality of specimens to be assayed for target substances of low concentrations, the trace quantities of constituents contained in one preprocessed specimen are left as a contaminant in the dispensing/mixing mechanisms or filtering mechanism of the preprocessing apparatus after the preprocessing of that specimen. This carry-over during the preprocessing of one specimen will reduce the accuracy of separation/extraction of the next specimen. This can be prevented by thoroughly cleaning the internal parts of the apparatus after the preprocessing of the previous specimen. Another method to prevent this problem is removing, for each specimen, parts that are likely to remain contaminated such as the dispensing pipettes, mixing parts, filtering parts, and others, and replacing these parts with exchangeable ones (hereinafter, called disposable parts). The other method is integrating these parts or using parts of common specifications to reduce the number of disposable parts.
Non-Patent Document 1 describes examples of preprocessing and separation/extraction techniques for specimens, the techniques required in measuring the concentration of an immunosuppressant drug in whole blood by means of liquid chromatography-mass spectrometry. According to Non-Patent Document 1, preprocessing and separation/extraction processes conducted prior to the measurement of liquid chromatography-mass spectrometry include:
(a) Hemolyzation (blood dissolving) by adding pure water or the like to whole blood,
(b) protein precipitation by adding zinc sulfate and/or methanol after the hemolyzation,
(c) centrifugation for removing the precipitated proteins, and
(d) solid-phase extraction of the supernatant cleared of the precipitated proteins, to further remove impurities likely to interfere with liquid chromatography-mass spectrometry.
As a method for removing protein sediments that do not use centrifugation, a method is also known where the solution is forcibly passed through a filter and the protein sediments stuck to the filter is removed. However, although individual apparatuses dedicated for the steps of preprocessing methods (a), (b), (c), (d) are generally well known, there is no apparatus that has fully automated steps (a) to (d).
For example, if the protein sediments in step (b) remain to be further processed, impurities will be redissolved from the sediments during the washing and eluting phases of solid-phase extraction step (d). The impurities will significantly deteriorate the accuracy of subsequent mass spectrometric measurement. It is therefore necessary to remove the sediments in step (c).
The operations spanning these steps are difficult to automate, and thus, these operations have traditionally been performed with manual operation. This has increased the number of containers/vessels and implements used for temporary collection of the solution between steps and for the dispensing operations in the next steps. Additionally, due to trace constituents sticking to these containers/vessels and implements, a carry-over as well as a loss in the collection, reduction in total analytical sensitivity and accuracy are caused, and therefore obstructing the analyzable volumes of specimens to become smaller.
Patent Document 1 relates to an example of a batch-processing type of solid-phase extraction technique as preprocessing for separating and extracting target constituents from specimens to be processed. In this conventional technique, a solid-phase extraction plate is formed with 96 solid-phase extraction columns for accommodating the specimens and is mounted in an upper vacuum rack, which is set on a horizontally and vertically movable mechanism. The upper vacuum rack is pressed against a lower vacuum rack, and each specimen is suctioned from the lower vacuum rack by a vacuum pump. Consequently, the target constituents are adsorbed onto a filter provided in each solid-phase extraction column, and the target constituents adsorbed onto the filter are eluted and extracted. Patent Document 1 also discloses that a low-carryover type disposable pipette/nozzle tip is used to dispense the specimen. Further, a method of mixing by suctioning/discharging solution with the disposable pipette/nozzle tip to achieve low-carryover mixing is disclosed.
The automated solid-phase extraction process according to Patent Document 1 is of a batch-processing type, so that efficient preprocessing is possible when the number of specimens to be preprocessed and assayed is an integral multiple of the number of specimens (96), which the solid-phase extraction plate can retain. The number of specimens to be actually preprocessed, however, is not always fixed. Using a fixed number of solid-phase extraction columns (96 pieces), as in the conventional technique, therefore, has usually caused some of the wells (solid-phase extraction columns) to remain unused. A decrease in efficiency was thus inevitable, where relative cost of analysis increased, and waste disposal volume increased.
In addition to the above-mentioned problem of deterioration in cost performance, when specimens are needed to be analyzed chronologically sequentially and randomly, a problem such that preprocessing of the next specimen can not be started until the earlier started preprocessing is completed occurs. That is, specimens can not be continuously loaded into the preprocessing apparatus. This leads to an increase in turn-around-time (TAT), a waiting time taken until analytical results are obtained.
Further, whereas the well plate that forms the large number of wells (96 pieces) allows simultaneous movement of the specimens accommodated in the wells, the well plate itself is of a relatively large size. Due to this, spaces are required for storage/retraction of the well plates, for operations such as dispensing, mixing, pressurization, and filtering, for installing the devices required for movement and various processing operations, and the like. As a result, reduction in the size of apparatuses has been obstructed.
Patent Documents 2 and 3 relate to known examples of an automatic analyzer, which conducts optical measurement after executing preprocessing for separation/extraction of target constituents by causing reactions with a reaction reagent that contains magnetic beads, and separating/extracting analytic molecules. The Patent Documents 2, 3 also discloses a technique relating to a disposable dispensing pipette used for preprocessing, for reducing carry-over. These conventional techniques, unlike the technique in Patent Document 1, employ sequential preprocessing, not batch-based preprocessing. Therefore, the disadvantages of the technique in Patent Document 1, that is, disadvantages appearing when a specific number of specimens (96 pieces) is not reached, such as the analytical cost increase, waste parts increase, and TAT increase due to incapability of continuous loading can be avoided. In these known techniques, however, as described in Patent Document 3, if dispensing/mixing is conducted so as to achieve minimum carry-over for each of a plurality of reaction steps in preprocessing, related parts need to be replaced with new disposable parts for each step of reactions, during a first reagent step and a second reagent step. This replacement has led to increases in the number of disposable parts consumed and the number of disposable parts to be retained by devices.